Response to ReviewersRevised Manuscript with Track ChangesManuscript

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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Reviewers' comments:

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods:

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Yes

Reviewer #2: "Minor Revision"

The methodology applied in this study is comprehensive and well-structured, incorporating a variety of techniques for the validation of results. However, I would require clarification on certain aspects, which I outline below:

Line 119. This paragraph is somewhat unclear, as it states that parasite maintenance is carried out in Wistar rats, while larval collection is performed in mice. Could you clarify the methodology?

Line 179. Was the inclusion of an adjuvant-only control group considered in the experimental design?

Line 187. As I understand it, the booster immunizations were consistently administered intramuscularly with rTsCLP; was revaccination with rAd5TsCLP via the intranasal route considered at any point?

Line 234. It should be noted that the mice were previously anesthetized before blood collection from the orbital venous plexus."

Line 249. Was the goblet cell count performed using Hematoxylin and Eosin staining, or was another more specific stain, such as Alcian blue, used?

Line 254. It would be appropriate to specify the methodology used for the recovery of adult worms from the intestine, as well as the procedure for obtaining muscle larvae.

Results:

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Yes

Reviewer #2: "The results align with the objectives of the study and are appropriately presented. However, the histological images, at least in the version of the manuscript reviewed, lack sufficient quality. Additionally, I would like to offer a couple of recommendations.

Line 377. Some studies suggest that Trichinella spiralis infection naturally induces an increase in goblet cell numbers; however, due to endoplasmic reticulum stress, these cells may be unable to adequately produce mucins, thereby compromising their protective role. In your study, I would recommend assessing mucin expression, as an increase in goblet cell numbers alone is not necessarily indicative of enhanced protection..

Line 410. Why were cervical lymph nodes analyzed instead of mesenteric lymph nodes?

Conclusions:

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Yes

Reviewer #2: (No Response)

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: Minor revision is recommended according to the suggestions provided in the previous sections.

Summary and General Comments:

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Reviewer comments on PNTD-D-25-00587

In this manuscript titled “Heterologous prime-boost immunization based on a human adenovirus 5 vectored containing Trichinella spiralis Cystatin-like protein elicits protective mucosal immunity in mice”(PNTD-D-25-00587), the authors dressed an important problem. The objectives of this study are clear and the experimental design is appropriate. Overall, I recommend that this manuscript could be accepted for publication after some minor revisions. They are as follows:

1. General comment: Ensure consistent use of italics for species or genus names (e.g., Trichinella spiralis,); Include in your description if you talk about larvae or adult worms (in the discussion thread as well). And the Latin names of the genus in the references, Salmonella typhimurium-> Salmonella typhimurium in italics.

2. Line 55: put a space before the square brackets [], such as larvae[1]-> larvae [1].

3. Line76. Provide a brief description of the basic structural features of adenovirus (e.g., capsid organization, genome structure) to contextualize the vector design for readers unfamiliar with adenoviral systems.

4. Line92-94. Previous published studies should be cited to support that cystatin-like proteins from Trichinella spiralis are localized in β-stichocytes and expressed in various developmental stages, including the early invasion and muscle larval stages (Tang et al., 2015; Guiliano et al., 2009).

a) Tang B, Liu M, Wang L, Yu S, Shi H, Boireau P, Cozma V, Wu X, Liu X. Characterisation of a high-frequency gene encoding a strongly antigenic cystatin-like protein from Trichinella spiralis at its early invasion stage. Parasit Vectors. 2015; 8:78. doi: 10.1186/s13071-015-0689-5.

b) Guiliano DB, Oksov Y, Lustigman S, Gounaris K, Selkirk ME. Characterisation of novel protein families secreted by muscle stage larvae of Trichinella spiralis. Int J Parasitol. 2009;39(5):515-24. doi: 10.1016/j.ijpara.2008.09.012. Epub 2008 Oct 21.

5. Line182. Please verify the selection of adjuvant CpG1018 over other adjuvants (e.g., its mechanism of action as a TLR9 agonist and relevance to mucosal immunity).

6. The authors should explicitly specify whether the control group received the same 250 muscle larvae as the infection group to ensure consistency in experimental design and rule out volume-dependent effects.

7. Line398. While cytokine profiling is comprehensive, the roles of IL-12p70, MCP-1, and IL-17 in the observed immune response should be explicitly presented in the results section.

8. Figure 6c. Correct the x-axis label "AdTsCLP:rTsCLP" as "rAd5TsCLP:rTsCLP" to align with the nomenclature used in the Methods section.

9. Suggest the authors to clarify whether the term 'Special sIgA' in Figure 4 refers to 'TsCLP-specific sIgA' and revise the figure accordingly for consistency with the legend, and to clearly state the statistical methods used in Figure 3, including the type of test and significance thresholds in the figure legend.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: 2025-5-6-Comments on PNTD-D-25-00587PNTD.docx

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Submitted filename: Questions.docx

Dear Dr Bai,

We are pleased to inform you that your manuscript 'Heterologous prime-boost immunization based on a human adenovirus 5 vectored containing Trichinella spiralis Cystatin-like protein elicits protective mucosal immunity in mice' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Javier Sotillo

Academic Editor

PLOS Neglected Tropical Diseases

Jong-Yil Chai

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The revised manuscript (PNTD-D-25-00587R1) has been obviously improved. All questions and errors have been highlighted and corrected. I recommend that this manuscript can be accepted for publication in the present form.

Reviewer #2: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: Yes:  Juan José García-Rodríguez